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A small peptide called FGL boosts learning and memory when administered to rodents, and is poised to begin clinical trials in Alzheimer’s disease patients this year. Intriguingly, FGL sharpens memory in wild-type rats as well as in several disease models. While prior studies suggested that the heightened learning resulted from improved synaptic plasticity in the hippocampus (see Dallérac et al., 2011), the mechanism was unclear. Now, in the February 21 PLoS Biology, researchers led by José Esteban at the Universidad Autónoma de Madrid, Spain, detail the signaling pathway behind this effect. They report that FGL treatment stimulates activity-dependent delivery of glutamate receptors to synapses, leading to a long-term enhancement of synaptic transmission.

Scientists contacted by Alzforum expressed enthusiasm for the findings. “The effect on synaptic transmission is impressive, and represents the only peptide that I know of that is capable of enhancing transmission and plasticity,” wrote Roberto Malinow at the University of California, San Diego.

Elisabeth Bock and colleagues at the University of Copenhagen, Denmark, designed the 15-amino-acid peptide in 2004, basing it on a portion of neural cell adhesion molecule (NCAM). NCAM is known to play a role in synaptic plasticity (see, e.g., Dityatev et al., 2000). Bock and colleagues showed that the FG loop (FGL) from the second fibronectin type III module of NCAM binds and activates fibroblast growth factor receptor 1 (FGFR1) (see Kiselyov et al., 2003). The FGL peptide mimics this activity and improves cell survival in primary neuronal cultures exposed to toxins, the authors reported (see Neiiendam et al., 2004). Moreover, FGL injected subcutaneously into wild-type rats crosses the blood-brain barrier and enhances several forms of memory, including fear conditioning, social and motor learning, and spatial memory tested in the Morris water maze (see Cambon et al., 2004; Secher et al., 2006). Enhanced spatial memory persists for as long as two weeks after peptide administration. Furthermore, treated rats behave normally in an open field test, suggesting the peptide does not increase anxiety. Behavioral and social problems often accompany changes in learning, said Philip Washbourne from the University of Oregon, Eugene.

Esteban and colleagues wanted to dissect the mechanisms behind FGL’s effects. Joint first authors Shira Knafo and César Venero treated hippocampal slice cultures with 10 μg/ml FGL for 24 hours, then removed FGL for a day before electrophysiological testing. Peptide treatment heightened AMPA receptor-mediated transmission at excitatory CA1 synapses, which the authors showed was due to insertion of additional AMPA receptors. Inhibiting protein kinase C (PKC), a downstream effector of the FGF1 receptor, eliminated the AMPA receptor influx. To demonstrate that the same mechanism occurs in vivo, the authors administered both FGL and the PKC inhibitor into rat brains through a cannula, and found that the inhibitor abolished FGL-mediated memory enhancement.

Importantly, enhanced synaptic transmission does not occur spontaneously after FGL treatment. Instead, the peptide seems to facilitate long-term potentiation (LTP) in response to synaptic activity. In FGL-treated hippocampal slices, electrical stimulation induced LTP nearly twice as strongly as in untreated slices, and inhibiting PKC prevented this effect. Furthermore, when the authors blocked NMDA receptors, which are crucial for LTP, FGL treatment no longer pumped up AMPA receptor delivery. Esteban notes that this activity dependence is critical for a cognitive enhancer. If the peptide indiscriminately increased synaptic transmission, it might overexcite neurons and lead to epilepsy, he said. But by heightening synaptic plasticity only in response to activity, the peptide helps the animal encode information more easily, leading to better memory.

“I find it intriguing that memory processes can be improved over normal levels,” Esteban told Alzforum. This implies that the human memory system is not running at the top of its possible performance, he said. Esteban pointed out that FGL recruits physiological memory mechanisms, which suggests that the peptide may have few side effects. It also may not need to be given chronically. Esteban and colleagues found that PKC, as well as other proteins involved in LTP, stays activated for at least 24 hours after FGL removal. The peptide therefore provides a long-lasting augmentation of synaptic plasticity, in agreement with in-vivo results. “We still don’t know what allows the system to stay in this sensitized state,” Esteban noted.

In addition to looking at electrophysiology, the authors examined the structure of dendritic spines in the CA1 region. However, they found no evidence that FGL increased spine density or changed spine shape. Previous studies have also reported no change in spine density after FGL treatment, although earlier work did show evidence of alterations in fine structure (see Popov et al., 2008).

“The synaptic plasticity mediated by the FGL agonist, therefore, would appear to relate to retuning the strength of existing cell synapses,” rather than to creating new synapses, Ciaran Regan at University College Dublin, Ireland, wrote to Alzforum. This implies the peptide may be most effective for treating conditions where synaptic transmission may be weakened or perturbed, for example, in depression, rather than disorders such as AD, where synapses are lost, Regan suggested (see full comment below).

Other commentators expressed enthusiasm about the therapeutic potential of the peptide, while noting some cautions. Washbourne pointed out that it will be important to do more extensive behavioral tests, in particular, looking for effects of FGL on social behavior, before taking the peptide to clinical trials. Paul Lombroso at Yale University, New Haven, Connecticut, suggested the peptide might have broad applicability for numerous types of cognitive dysfunction, telling Alzforum, “I think this is an excellent paper, and very exciting in terms of our attempts to find reagents that may improve cognitive deficits.”

Does the peptide hold potential for treating Alzheimer’s disease? One published study from Bock’s group suggests it does. Rats injected with Aβ oligomers develop signs of AD-like neuropathology and failing memory, but when also given FGL, either subcutaneously or intranasally, these deficits do not develop. In addition, Aβ-injected rats having more advanced pathology improved after FGL treatment, showing fewer amyloid plaques and better memory than untreated controls (see Klementiev et al., 2007). Experiments in rats, dogs, and monkeys found no toxic effects from the peptide, and an eight-day human study on 24 healthy male volunteers revealed no ill effects from a single dose of FGL given intranasally (see Anand et al., 2007). Based on these data, Enkam Pharmaceuticals, a biotechnology company based in Copenhagen, Denmark, plans to begin clinical trials of a modified form of FGL in 2012 (see company press release). In cooperation with a consortium of companies, universities, and AD patient groups, Enkam will lead three Phase 1 safety studies, one Phase 2a study in AD patients, and also a pilot study in stroke patients. The consortium has been awarded a €6 million grant from the European Union’s Seventh Framework Programme to conduct these studies (see also ARF related news story).—Madolyn Bowman Rogers

Comments

These studies explore the cognition-enhancing actions of a peptide agonist of the neural cell adhesion molecule (NCAM), the FG loop (FGL) peptide, synthesized from the second fibronectin type III module of NCAM. The identification of FGL as a cognition-enhancing agent is well established in the literature, and this new and most comprehensive dataset now provides further information on the molecular mechanisms by which this peptide may mediate its action. The bottom line outcome of these studies is that FGL enhances cognition in rodents and long-term potentiation (LTP), a cellular model of memory and learning, in hippocampal slices. This facilitation is demonstrated to be mediated by FGL enhancing the delivery of AMPA-type glutamate receptor into excitatory synapses following activation of NMDA-type glutamate receptors. Importantly, these effects are reported to be specifically mediated by PKC activation.

This new dataset opens up pathways (literally) to further experiments such as the effect of the FGL peptide on the polysialylation status of NCAM, which also regulates plasticity and learning through modulation of NMDA receptor signaling using the GluN2B-Ras-GRF1-p38 MAPK pathway (Kochlamazashvili et al., 2010). Understanding crosstalk, if any, between these two pathways may be crucial to revealing core neuroplastic deficits in Alzheimer’s disease, in which postmortem studies have shown NCAM polysialylation to be significantly increased in the hippocampal formation (Mikkonen et al., 1999).

Secondly, it is of particular interest that, despite a most rigorous morphological analysis, the FGL peptide fails to have any impact on synapse density in the CA1 region of the hippocampus. This finding is consistent with previous studies on the effect of FGL on plasticity in the dentate and CA3 regions of the hippocampus (Popov et al., 2008; Ojo et al., 2011)—as an aside, the dentate study found an increase in the frequency of coated pits, which would be consistent with the AMPA receptor trafficking data reported in this PLoS Biology paper. The synaptic plasticity mediated by the FGL agonist, therefore, would appear to relate to retuning the strength of existing cell synapses in which the learning and memory networks are distinguished by the composition of the cells that are coactivated. This contrasts with the synapse assembly mechanism in which the creation and pruning of synapses elaborates a network of specific groups of novel synapses with a connectivity scheme that has been optimized for each response (Ziv and Ahissar, 2009).

As a consequence, the nature of the synaptic plasticity wrought by novel drug strategies, such as that of the FGL peptide, may have significant implications for their future clinical development. Drugs that augment synapse elaboration mechanisms, for example, may have greater impact on neurodegenerative conditions such as Alzheimer’s disease, in which neuron loss and synapse loss provide the neuropathological correlative for dementia (DeKosky and Scheff, 1990). The FGL peptide agonist, which is known to be well tolerated in humans (Anand et al., 2007), primarily appears to retune the strength of existing cell synapses and, as such, may have more relevant neurotherapeutic potential in conditions other than Alzheimer’s disease. A role for FGL in the treatment of depression has been previously suggested by the authors (Aonurm-Helm et al., 2008).